Ch. 13 - DNA Flashcards
Griffith’s experiment
In 1928, when Griffith inoculated mice with a mixture of living R-type (rough type - nonvirulent) bacterial cells and heat-killed S-type (smooth type - virulent) bacterial cells, the mice died of pneumonia; Griffith concluded that in the presence of dead S-type pneumococcus cells, some of the living R-type cells has been transformed into virulent S-cells (transforming factor = DNA)
Avery, McLeod, McCarty experiment
In 1944, Avery et al conducted experiments with the Streptococcus pneumoniae bacteria where protein, RNA and DNA were eliminated from the heat-killed S-type cells; these samples were mixed with live R-type cells and when the cells were treated with DNase (breakdown of DNA), the transforming activity was lost and R-type cells were not transformed into S-type cells; concluded that DNA is the transforming factor
Hershey-Chase experiment
Used bacteriophage T2 virus to determine whether DNA or protein is genetic material; viruses were labeled with radioactive sulfur and radioactive phosphorus and mixed with bacteria; the bacteria were isolated and found to contain radioactive phosphorus; determined that DNA transferred into the bacteria and was responsible for redirecting the genetic program of the bacterial cell
Rosalind Franklin
Used X-ray diffraction to visualize crystallized DNA; her data suggested that DNA is structured as a double-stranded helix; 10 nucleotides per full turn; 3.4 nm in length; diameter of 2 nm suggested that the sugar-phosphate backbone of each DNA strand must be on the outside of the helix
Chargaff’s rule
In any DNA sample, the amount of purines equal the amount of pyrimidines, the amount of adenine equal the amount of thymine (A=T) and the amount of guanine equals the amount of cytosine (G=C); with a DNA sample, you can identify the percentage of the nitrogenous bases in the sample (ie. 10% adenine = 10% thymine, 40% guanine = 40% cytosine)
Watson and Crick’s model
In 1953, Francis Crick and James Watson used model building to solve the structure of DNA: their model had the nucleotide bases on the interior of the two strands, with a sugar-phosphate “backbone” on the outside, the two DNA strands ran in opposite directions or antiparallel and that bases were purine-pyrimidine pairs
DNA’s four key features
1) DNA is a double-stranded helix 2) DNA is usually a right-handed helix 3) DNA is antiparallel 4) DNA has major and minor grooves (major grooves are exposed to allow DNA binding proteins to attach and replicate or transcribe)
Antiparallel strands
DNA replicates and grows in a 5’ to 3’ direction; both strands run in opposite directions
Components of DNA replication in a test tube
Deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP (dNTPs are monomers of DNA); DNA template; DNA polymerase; salts and pH buffer
Base Exposure in the Grooves
The surface of the A-T and the C-G base pairs chemically distinct, allowing other molecules such as proteins to recognize specific base pair sequences and bind to them; the binding of proteins to specific base pair sequences is the key to protein-DNA interactions necessary for replication and expression of genetic information in DNA
Semi-conservative replication
Each parent strand serves as a template for a new strand; two new DNA molecules each have one old and one new strand
Conservative replication
Two DNA molecules are formed, one parental strand and one new strand
Dispersive replication
Fragments of the original DNA molecule serve as template for assembling two new molecules, each containing old and new parts, perhaps at random
Meselson and Stahl Experiment
Experiment demonstrated that DNA replication is semiconservative; used density labeling to distinguish between parent strands of DNA and newly copied ones; DNA was extracted and after two replication cycles, two bands of DNA were seen, one of intermediate density and one of light density. This result is exactly what the semiconservative model predicts: half should be 15N-14N intermediate density DNA and half should be 14N-14N light density DNA
Requirements for DNA replication
DNA template, dNTPs, helicase, topoisomerase, single strand binding proteins (SSBs), RNA primase, DNA polymerase III, RNase H, DNA polymerase I, DNA ligase
Replisome
Complex molecular machine that carries out DNA replication; forms two new double stranded DNA sequences; remains stationary while DNA moves through the complex; includes a helicase, a polymerase and a sliding clamp; the replisome assembles at a region of the DNA, called the replication fork, where the double-stranded DNA is separated into two individual strands, which are both subsequently copied in the 5′ to 3′ direction of the DNA
DNA replication
The pre-replication complex binds to the ori (origin point) and helicase starts to unzip the double-stranded helix forming a replication fork; single-strand binding proteins coat the separated strands of DNA near the replication fork, keeping them from coming back together into a double helix; primers are short starter strands that are synthesized by RNA primase and are placed at the start of the new strand; DNA polymerase III then adds nucleotides to the 3’ end of the primer until replication of that section of DNA is complete; RNAase H removes the RNA primer, DNA pol I replaces the RNA primer with DNA, DNA ligase catalyzes the formation of the final phosphodiester linkage of the backbone
DNA polymerase structure
Large protein enzyme that is shaped like an open right hand with a palm, thumb and fingers; the palm is the active site of the enzyme that brings together the dNTPs with the DNA template; the finger region has precise shapes that recognize the different nucleotide bases (dNTPs)
Topoisomerase
This enzyme prevents the DNA double helix ahead of the replication fork from getting too tightly wound as the DNA is opened up. It acts by making temporary nicks in the helix to release the tension, then sealing the nicks to avoid permanent damage
Leading strand
During DNA replication, at the replication fork, the leading strand grows at the 3’ end as the fork opens
Lagging strand
During DNA replication, the lagging strand is oriented so that as the fork opens up, its exposed 3’ end gets farther and farther away from the fork; this strand is made in fragments because, as the fork moves forward, the DNA polymerase (which is moving away from the fork) must come off and reattach on the newly exposed DNA
Okazaki fragment
While the leading strand grows continuously forward, the lagging strand grows in shorter, “backward” stretches with gaps in between them; each fragment requires its own primer synthesized by RNA primase;
Sliding DNA clamp
A protein that keeps the polymerase-DNA complex stabilized and in close contact; prevents DNA polymerase from dissociating from the DNA template; the clamp increases the efficiency of polymerization by keeping the enzyme (DNA pol) bound to the substrate
Telomere
Eukaryote chromosomes have repetitive sequences (~2500 times) at the ends; on lagging strand, when the terminal Okazaki primer is removed, no DNA can be synthesized to replace it; at the end of the chromosome, however, there is no neighboring Okazaki fragment to provide the needed hydroxyl to start the DNA replacement, resulting in incomplete replication of the chromosome end; this short piece of DNA is removed; chromosome becomes shorter w/each replication; after many divisions, genes may be lost and cell dies
Telomerase
Continuously dividing cells (ie. bone marrow stem cells) have telomerase, an enzyme that catalyzes addition of lost telomeres; typically expressed in most cancer cells which allow the cells to keep dividing
DNA repair
1) Proofreading: as DNA polymerase adds a nucleotide to a growing stand, it recognizes mismatched pairs and replaces the base 2) Mismatch repair: newly replicated DNA is scanned for mistakes by other proteins and the protein complex corrects the mismatch 3) Excision repair: NER enzymes constantly scan DNA for damaged bases, which are excised and DNA pol I adds the replacement bases
Xeroderma pigmentosum
Genetic disorder that lacks an excision repair mechanism that repairs damage caused by UV radiation; most commonly due to a defect in a NER gene; thymine dimers accumulate in skin cells which interferes with base pairing during replication
